Plants receive environmental stresses, such as salt, dehydration, high temperature, low temperature, intense light, air pollution, and the like. Salt damage and dehydration are the most problematic stresses from the viewpoint of agricultural production. Salt damage occurs not only in areas that originally have a high salt content but also in farmland due to irrigation, which was problem-free in the past. At present, agricultural lands over as large an area as 12,000,000 hectares suffer damage due to salt or drought in Asia, and agricultural lands over as large an area as 9,500,000 hectares actually remain unused due to salt damage. In particular, rice is a staple grain in Asia. If salt stress tolerance could be imparted to rice, accordingly, unused farmland could be converted for food production. This would positively affect the stabilization of grain production in the world. Up to the present, a variety of attempts have been made in order to produce environmental stress tolerant plants that can be cultivated under bad environmental conditions or in poor soil via gene recombinant technologies. For example, a gene, the expression of which is induced by salt stress, may be isolated and then allowed to express, thereby producing salt tolerant plants. Examples of known genes that impart salt stress tolerance include the betaine synthase gene derived from manila grass (Zoysia matrella) (JP Patent Publication (Unexamined) No. 2001-309789), the choline oxidase gene derived from Arthrobacter globiformis (Mohanty A., et al., Theor. Appl. Genet., 106, pp. 51-57, 2002), the chloroplast glutamine synthase derived from rice (Hoshida H., et al., Plant Mol. Biol. 43, pp. 103-111, 2000), the active transcription factor derived from rice (OSDREB; Dobouzet J. G., et al., Plant J. 33, pp. 751-763, 2003), and the Na+/H+ antiporter gene derived from Atriplex gmelinii var. spontanea (JP Patent Publication (Unexamined) No. 2000-157287). Among the enzyme genes associated with sugar metabolism or synthesis, the genes associated with trehalose synthesis derived from E. coli (Jang I. C., et al., Plant Physiol., 131, pp. 516-524, 2003) and the galactinol synthase (AtGloS) genes that synthesize galactinol from UDP galactose have been reported to be associated with impartation of water stress tolerance such as dehydration, salt, or low temperature stress to Arabidopsis thaliana (“Saibou Kougaku (Cell Technology),” vol. 21, No. 12, pp. 1455-1459, 2002; Teruaki T. et al., The Plant Journal, 29 (4), pp. 417-426, 2002). In the case of dicotyledonous plants, there has been an example wherein the salt stress tolerance of plants has been evaluated with a salt concentration of 200 mM over a period of 10 weeks in tomatoes transformed with the Na+/H+ antiporter genes derived from Arabidopsis thaliana (Zhang H. X., Blumwald E., Nature Biotechnol., 19, pp. 765-768, 2001) or in Brassica plants (Zhang H. X. et al., Proc. Natl. Acad. Sci. U.S.A., 98, pp. 12832-12836, 2001), and harvests of fruits or seeds have been reported. In the case of monocotyledonous transgenic plants, however, there has been no gene that imparted salt stress for a long period of time from transplantation to seed harvesting. For example, transgenic rice exhibits salt stress tolerance for only a short period of time of 13 days at 100 mM, 2 weeks at 150 mM, and 3 days at 300 mM. Accordingly, it is difficult to consider that the aforementioned genes could impart salt tolerance to plants that would be efficient for the actual process of production over a period of from several weeks to several months, from seedling transplantation to seed harvesting, under the present circumstances.
UDP-glucose 4-epimerase is an enzyme that catalyzes bilateral reactions from UDP glucose to UDP galactose and vice versa. Up to the present, the plant-derived UDP-glucose 4-epimerase gene (hereafter it may be referred to as the “UGE gene”) has been isolated from, for example, Arabidopsis thaliana or guar (Reiter W. D., Vanzin G. F., Plant Mol. Biol., 47, pp. 95-113, 2001). However, there has been no UGE gene that is known to be capable of imparting salt stress tolerance. Also, no plant species wherein the expression of UGE genes is induced by salt stress has been reported.
Galactose inhibits growth at the shoot in the process of gemmation of the dicotyledonous plant Arabidopsis thaliana. This is considered to result from accumulation of UDP-galactose or galactose-1-phosphoric acid because the plant could not use up externally provided galactose. In the case of plants into which the 35S and nosT expression cassettes of the UGE genes of Arabidopsis thaliana have been introduced, it is reported that growth is unlikely to be inhibited even in the presence of galactose. Also, the availability of the UGE gene as a selection marker for a transgenic plant has been pointed out (Reiter W. D., Vanzin G. F., Plant Mol. Biol., 47, pp. 95-113, 2001; Dormann P. and Benning, C., the Plant Journal, 13, pp. 641-652, 1998). It is considered that the growth of the plants into which UGE genes have been introduced is not inhibited in the presence of galactose, because the UGE genes convert the accumulated UDP-galactose to UDP-glucose. In contrast, it is reported that galactose has the effect of inhibiting the stretching growth of some tissues of seedlings, coleoptiles or sheath leaves, and seminal roots by phytohormones, such as auxin or gibberellin, of monocotyledonous plants (Inouhe M., et al., Physiologia Plantalum, 66, pp. 370-376, 1986); however, there has been no report or research on the influence thereof on physiological phenomena associated with tissue culturing, such as rooting. There have been no reports of any experiments, whereby the growth of monocotyledonous plants, including grass, in the presence of galactose is inspected by introducing the UGE genes therein.
Further, antibiotic tolerant genes, such as kanamycin tolerant genes or hygromycin tolerant genes, remain in genetically engineered organisms (GMO). This is the most serious issue of concern in recent years, in terms of the safety of GMO. Such genes are referred to as selection markers (marker genes) for screening the cells into which the target genes have been effectively introduced at an early stage. They become unnecessary after regeneration of plants from cells, rooting, and acclimation. In contrast, a method of selection that involves the use of sugar, which is considered to impose a slighter influence on human bodies, has been reported in recent years. In this method, the sugar isomerase genes of microorganisms are employed as markers, and selection can be made with the use of xylose (Haaldrup A., et al., Plant Cell Reports, 18, pp. 76-81, 1998) or mannose (Joersbo M., et al., Molecular Breeding, 4, pp. 111-117, 1998). These marker genes are, however, derived from microorganisms, and the safety of their DNA cannot be perfectly ensured, since humans have not ever ingested such DNA as food. Therefore, the development of highly safe selection markers that can serve as alternatives to antibiotic tolerant genes and expression vectors using the same has been awaited.
Accordingly, an object of the present invention is to provide a novel gene that can impart salt stress tolerance to plants for a long period of time and salt stress-tolerant transgenic plants into which such gene has been introduced. It is another object of the present invention to provide highly safe selection markers that can serve as alternatives to antibiotic tolerant genes.